One important issue for the effective containment and control of any agent is a timely and accurate diagnosis of patients infected with that agent. One aspect of such a diagnosis includes differential diagnosis. By differential diagnosis it is meant the determination and identification of those patients having a disease state or condition caused by a disease agent of interest, and those patients having a disease state condition caused by one or more secondary agents that result in a similar clinical manifestation. This differential diagnosis is critical since the symptoms/clinical manifestations observed in conditions caused by the disease agent of interest may also be observed in conditions caused by the secondary agents. Therefore, the possibility of misdiagnosis is a significant issue. Such misdiagnosis can result in both false positives, and false negatives. Each of these types of misdiagnosis has a detrimental impact of the containment and control of the disease agent. For example, a false negative will allow an infected individual to continue to spread the disease agent to the general population. A false positive will result in an increased burden on the healthcare system and on the individuals required to undergo needless treatment.
False positives and false negatives can be of special concern in cases of bioterrorism, where accurate and rapid identification of the causative agent is required for containment, control and an effective public health response. The growing concern regarding the use of bioterrorism has prompted Federal health agencies to accelerate measures to protect the public from such attacks. In February, 2002, the National Institute of Allergy and Infectious Diseases (NIAID) released its Biodefense Research Agenda for CDC category A, B and C agents. One of the goals of this research agenda was the development of diagnostic test applicable to agents that may be used in bioterrorism. The Agenda stated “A successful response to a bioterrorist threat requires diagnostics that can identify the pathogen involved. However, the initial clinical signs and symptoms of many agents considered biothreats are nonspecific and resemble those of common infections. The ability to rapidly identify the introduction of a bioterrorism organism or toxin will require diagnostic tools that are highly sensitive, specific inexpensive, easy to use, and located in primary care settings.”
As an example of the problems and issues discussed above, consider the recent outbreak of the severe acute respiratory syndrome (SARS). The clinical symptoms of SARS, especially in the early stages, included fever, chills and moderate to severe coughing. Obviously, these symptoms are observed in a number of conditions caused by other agents. Other agents capable of causing conditions with SARS-like symptoms include, but are not limited to, respiratory syncytial virus, parainfluenzaviruses type 1 and type 3, influenza A and B viruses, enterovirus, adenovirus, Mycoplasma pneumoniae, and Chlamydia pneumoniae. 
There are three classes of diagnostic tests commonly used in the detection of disease agents: i) ELISA tests; ii) cell culture methods; and iii) molecular tests. Each of these tests has their own advantages and disadvantages. The ELISA (Enzyme Linked Immunoabsorbant Assay) is an antibody test. It detects antibodies to the disease agent in the serum of patients reliably by day 21 after the onset of clinical symptoms. ELISA is specific, but the detection comes too late for detection to be useful in disease management. It can not provide the much needed early information required for the containment and control of the disease agent.
Cell culture methods detect the presence of live agent. A sample is taken from an individual suspected of being infected with the disease agent and the disease agent is propagated in cultured cells or cultured according to defined conditions on selective culture media. Either process is a time-consuming, demanding and dangerous task, but it is the only means to show the existence of the live agent. As with the ELISA, the test is relatively specific, but the detection comes too late for detection to be useful in disease management.
The molecular tests generally use any one of a number of variations on the polymerase chain reaction (PCR). PCR can detect genetic material of an agent in various specimens (blood, stool, respiratory secretions or body tissue) from the individual suspected of being infected by the disease agent. Existing PCR tests are very specific, but lack sensitivity. This is because the agent may not yet be present in the patient specimens or the amplification and detection schemes fail to identify the genetic material. Therefore, a negative test can't rule out the presence of the disease agent in an individual.
Multiplex PCR allows the amplification of target sequences from multiple organisms in one reaction using multiple sets of locus specific primers. Therefore, multiplex PCR is suited to differential diagnosis. However, multiplex PCR methods have limitations. There are two major problems associated with the multiplex PCR method. One is that each target sequence (or locus) to be amplified has its own amplification efficiency. The locus specific amplification efficiency is determined by multiple factors including the composition of the primer targets, binding affinity of the primers to their targets, priming efficiency of the primers and availability of reaction components. Combining multiple target loci in one reaction may introduce incompatibility between various primer sets which results in preferential amplification or inhibition of some amplification reactions. The second issue is the identification of the optimal primer to locus ratio. If the primer concentrations are set too high, primer dimmers and background amplification will occur. If, however, the primer concentrations are too low, the desired exponential amplification of the target sequence will not occur.
In order to optimize multiplex PCR, the concentrations of primers, buffer, dNTPs, enzyme, and MgCl2 need to be determined empirically for each set of primer combinations. It is a time consuming process which needs to be conducted for each lot of the produced assay. A successful multiplex PCR is not guaranteed even after exhaustive optimization experiments.
The present disclosure provides a quick, accurate molecular diagnostic method for the diagnosis of a disease agent and/or the differential diagnosis of a disease agents in the presence of one or more secondary disease agents. Briefly nucleic acid samples are obtained from samples suspected of containing the disease agent and/or secondary disease agents; the nucleic acid may be DNA or RNA (either positive strand or negative strand) or a combination thereof. A multiplex amplification reaction is used to amplify pre-determined target sequences from the nucleic acid through one amplification reaction in one vial. The amplification products containing the target sequences are detected and differentiated using a multiplex detection strategy. The detection of the target sequence from a disease agent or secondary disease agent indicates its presence in the sample. Using the method disclosed herein, a diagnosis or differential diagnosis of a disease agent can be made in as little as 3 hours. The high throughput ability allows the analysis of hundreds of samples per day without the need for complex and time consuming optimization procedures for each primer combination. Such methods are lacking in the art.